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We report a new experimental method of direct selection, identification, and mapping of potential enhancer sequences within extended stretches of genomic DNA. The method allows simultaneous cloning of a quantity of sequences instead of tedious screening of the separate ones, thus providing a robust and high-throughput approach to the mapping of enhancers. The selection procedure is based on the ability of such sequences to activate a minimal promoter that drives expression of a selective gene. To this end a mixture of short DNA fragments derived from the segment of interest was cloned in a retroviral vector containing the neomycin phosphotransferase II gene under control of a cytomegalovirus (CMV) minimal promoter. The pool of retroviruses obtained was used to infect HeLa cells and then to select neomycin-resistant colonies containing constructs with enhancer-like sequences. The pool of the genomic fragments was rescued by PCR and cloned, forming a library of the potential enhancers. Fifteen enhancer-like fragments were selected from 1-Mb human genome locus, and enhancer activity of 13 of them was verified in a transient transfection reporter gene assay. The sequences selected were found to be predominantly located near 5' regions of genes or within gene introns. 相似文献
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The results of contextual analysis of 25 different middle repetitive DNA sequences are presented. It was shown that each of these repetitive DNA sequences contains at least one enhancer-like structure homologous to real enhancers, as well as to their consensus. The enhancer-like structures have been also revealed in the replication origin of some prokaryote genomes. The results are discussed in the light of a possible role of middle repetitive DNA sequences in the modulation of gene expression. Some aspects of genomes' evolution, in relation to enhancers, are also considered. 相似文献
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DNA topoisomerase II cleaves at specific sites in the 5'' flanking region of c-fos proto-oncogenes in vitro. 总被引:6,自引:0,他引:6
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We have analyzed 1 kb of cloned human c-fos sequence (-711 to +287) for calf thymus DNA topoisomerase II cleavage sites in vitro. Using the anti-tumor drug VP16 (demethylepipodophyllotoxin-beta-D-glucoside) with purified topoisomerase II, we identify twelve sites. Five sites are clustered around position -306 in a region that possesses enhancer-like properties. A second cluster of three sites is positioned 15 bp upstream of the TATA promoter element. With a HeLa nuclear extract as a source of topoisomerase II, a subset of cleavage sites is conserved within the two clusters. The cleavage sites in the enhancer-like element are conserved in the homologous region of the murine c-fos. These findings raise the possibility that topoisomerase II is involved in mediation of mitogen-induced c-fos expression. 相似文献
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Eukaryotic genomes are packaged into chromatin, where diverse histone modifications can demarcate chromatin domains that facilitate or block gene expression. While silent chromatin has been associated with long noncoding RNAs (lncRNAs) for some time, new studies suggest that noncoding RNAs also modulate the active chromatin state. Divergent, antisense, and enhancer-like intergenic noncoding RNAs can either activate or repress gene expression by altering histone H3 lysine 4 methylation. An emerging class of enhancer-like lncRNAs may link chromosome structure to chromatin state and establish active chromatin domains. The confluence of several new technologies promises to rapidly expand this fascinating topic of investigation. 相似文献
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Grzeskowiak R Amin J Oetjen E Knepel W 《The Journal of biological chemistry》2000,275(39):30037-30045
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The nucleotide sequence of a 784-bp segment of cloned caerulein mRNA obtained from the skin of Xenopus laevis was determined. It codes for five heterogeneous procaerulein peptides interspersed with three 147-bp intercaerulein segments (ICS). The ICSs contain six inverted repeats and five eukaryotic enhancer-like sequences. Evidence for the presence of multiple forms of caerulein mRNA is presented. 相似文献
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Action at a distance in Mu DNA transposition: an enhancer-like element is the site of action of supercoiling relief activity by integration host factor (IHF). 总被引:20,自引:6,他引:14
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The first committed step in the in vitro strand transfer reaction of a mini-Mu donor molecule is the formation of a Type 1 complex in which the Mu ends are held together in a non-covalent protein-DNA complex. Efficient formation of this complex at high levels of donor supercoiling (sigma approximately -0.06) requires the Mu A and Escherichia coli HU proteins. At in vivo levels of supercoiling, efficient reaction also requires E. coli integration host factor (IHF). We demonstrate that this supercoiling relief activity of IHF is mediated through an IHF binding site in the Mu early promoter region. This site is part of a larger enhancer-like element which includes operator 1 (01) and part of operator 2 (02) with the IHF site in between. The enhancer-like element stimulates the initial rate of the in vitro reaction 100-fold and acts in a distance-independent fashion. Inversion of the orientation of the element results in a total loss of enhancer activity in the absence of IHF. However, a 10-fold stimulation in the initial rate of reaction is induced by the addition of IHF. Furthermore, correct helical phasing between 01 and 02 is required for maximal activity. The results indicate that a specific geometrical configuration of the enhancer-like element, which includes a sharp bend between 01 and 02, is required for optimal induction of synapsis. 相似文献
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Detailed analysis of the mouse H-2Kb promoter: enhancer-like sequences and their role in the regulation of class I gene expression 总被引:76,自引:0,他引:76
Sequencing and deletion analyses of the H-2Kb promoter have suggested that several regions may be important for expression and regulation of this gene. Two of these regions are conserved inside the promoter of several genes coding for classical transplantation antigens, but not in the promoter of class I genes located in the Qa region. They display enhancer-like activity in cells that express H-2 genes, but show some tissue specificity in that they function very poorly in undifferentiated embryonal carcinoma cells in which H-2 genes are not expressed. They also have been shown not to be the target of the adenovirus-12 induced repression of class I gene expression recently demonstrated by Schrier et al. The promoter of the beta 2-microglobulin gene also contains a sequence with enhancer-like activity, but shares no homology with the H-2Kb promoter region. 相似文献
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Kazuko Aoyagi Cris Kuhlemeier Nam-Hai Chua 《Molecular & general genetics : MGG》1988,213(2-3):179-185
Summary The pea rbcS-3A gene codes for the small subunit of ribulose-1,5-bisphosphate carboxylase and its expression is regulated by light, tissue type and stage of development. Analysis in transgenic tobacco plants has shown that the upstream region contains an enhancer-like element which can confer light-regulated and organ-specific expression upon a reporter gene (Fluhr et al. 1986a). Here we address the question of whether the enhancer specifies not only organ specificity, but also expression in the correct cell types. In situ immunofluorescence and microdissection were used on transgenic tobacco plants containing the rbcS-3A enhancer fused to a reporter gene consisting of the cauliflower mosaic virus 35S promoter and the bacterial gene encoding chloramphenicol acetyltransferase (CAT). CAT levels are high in leaf mesophyll cells, but in the epidermis expression is restricted to guard cells. In the midrib, of the leaf and in the stem, there is considerable signal in the chlorenchyma and in the phloem region. This pattern of expression closely correlates with the distribution of the endogenous RbcS polypeptides and with the presence of chlorophyll. Our results demonstrate that the rbcS-3A enhancer-like element possesses all the necessary DNA sequences for expression in the correct cell types. 相似文献
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阐明基因转录调控机理一直是分子生物学的研究热点。增强子是广泛存在于真核、病毒及原核生物中的重要的调控元件之一,它通过与各种调控蛋白因子相互作用而发挥其转录增强调节功能。 原核转录增强子的发现是近几年的事,有关研究报道远少于真核和病毒增强子。1989年,潘卫、吴 相似文献
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Control of rDNA replication in Tetrahymena involves a cis-acting upstream repeat of a promoter element 总被引:30,自引:0,他引:30
A novel genetic scheme was used to isolate mutants altered in the formation or maintenance of amplified rDNA in the Tetrahymena macronucleus. One such mutant had a cis-acting rDNA mutation that affected the ability of mutant rDNA molecules to replicate in macronuclei in the presence of a wild-type (B strain) rDNA. The mutant rDNA was lost from these heterozygous macronuclei during vegetative cell divisions, although it was maintained normally in the homozygous or hemizygous state. In contrast, wild-type macronuclear rDNA of the C3 strain used to obtain the mutant outreplicated B strain rDNA in B/C3 heterozygote macronuclei. Sequence differences were found between wild-type B and C3 and mutant C3 rDNAs in the replication origin region, changing an upstream repeat of a highly conserved rRNA promoter element. We propose that the various rDNA alleles differentially compete for limiting amounts of trans-acting factors that bind to these enhancer-like repeats and positively regulate rDNA replication. 相似文献
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《Journal of molecular biology》2019,431(1):111-121
Cell-type specific gene expression programs are tightly linked to epigenetic modifications on DNA and histone proteins. Here, we used a novel CRISPR-based epigenome editing approach to control gene expression spatially and temporally. We show that targeting dCas9–p300 complex to distal non-regulatory genomic regions reprograms the chromatin state of these regions into enhancer-like elements. Notably, through controlling the spatial distance of these induced enhancers (i-Enhancer) to the promoter, the gene expression amplitude can be tightly regulated. To better control the temporal persistence of induced gene expression, we integrated the auxin-inducible degron technology with CRISPR tools. This approach allows rapid depletion of the dCas9-fused epigenome modifier complex from the target site and enables temporal control over gene expression regulation. Using this tool, we investigated the temporal persistence of a locally edited epigenetic mark and its functional consequences. The tools and approaches presented here will allow novel insights into the mechanism of epigenetic memory and gene regulation from distal regulatory sites. 相似文献